Pseudomonas aeruginosa is an increasing cause of life-threatening nosocomial respiratory infection and portends a worse outcome in bronchiectasis. Clinically, the host response and outcome is dependent on the initial stages of P. aeruginosa infection however, the molecular events that occur as a result of early bacteria-epithelial cell interactions remain unclear. The apical membrane of airway epithelial cells is the initial site of contact with organisms and plays an essential barrier role achieved through a highly organized array of apical membrane proteins linked to cytoskeletal scaffolding proteins. The focus of the proposed studies is to understand the molecular requirements and cellular consequences of P. aeruginosa contact-dependent exotoxin on airway epithelial cell apical membrane proteins during early bacteria-epithelial cell interaction. We have found that apical membrane organization required for host defense is dependent on forkhead factor foxj1 and Rho-mediated activity and can be disrupted by P. aeruginosa type III exotoxin ExoS. Following P. aeruginosa infection of primary culture differentiated airway epithelial cells, we observed a decrease in foxj1 expression followed by a change in epithelial cell phenotype consistent with the foxj1 deficient state. This was characterized by decreased apical localization of cytoskeletal scaffolding ERM family and PDZ-domain NHERF/EBP50 proteins and disrupted cilia basal bodies. We hypothesize that (1) the airway epithelial cell responses to P. aeruginosa ExoS disrupt apical membrane organization and (2) these effects of ExoS are mediated by loss of foxj1 expression and interruption of RhoA pathways to alter the cytoskeletal proteins of the apical membrane and impair host defense. The hypothesis will be tested using an integrated approach in a high fidelity mouse airway epithelial cell primary culture model and in vivo infection models to reveal cellular and molecular events in infection pathogenesis. Specific Aims are to 1) characterize the role of P. aeruginosa ExoS on apical membrane scaffolding proteins and molecules that regulate apical membrane organization, 2) determine the physiologic consequences of ExoS in vitro by assay of specific apical membrane functions of airway epithelial cells and in vivo using genetically deficient mouse models to evaluate epithelial cell-specific defects in host defense, and 3) investigate pharmacologic and genetic augmentation of molecules in apical membrane pathways to abrogate effects of exoS. The proposed studies will identify targets of P. aeruginosa-induced disruption of host defense and lead to new strategies for prevention of P. aeruginosa airways infection.